Prostate-specific membrane antigen (PSMA) targeting peptides

ABSTRACT

Described herein is the discovery of novel PSMA-specific peptides, which were identified through a novel combinatorial biopanning method. One of the novel PSMA-specific peptides discovered, GTIQPYPFSWGY (or GTI) (SEQ ID NO: 2), exhibits high binding affinity and selectivity to PSMA and PSMA-positive prostate cancer cells. It was found that GTI can mediate internalization of the apoptotic KLA peptide (SEQ ID NO: 10) to PSMA-positive LNCaP cells and induce cell death. Moreover, a FAM-labeled GTI peptide shows a high and specific tumor uptake in nude mice bearing human prostate cancer xenografts. It was demonstrated that the GTI peptide can be employed as a PSMA-specific ligand for prostate cancer diagnosis and/or for targeted drug delivery to prostate cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC § 119(e) to U.S.Provisional Application Ser. No. 62/297,593, filed on Feb. 19, 2016, theentire disclosure of which is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No.1R01AA021510 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 28, 2017, isnamed 745-13517_SL.txt and is 6,036 bytes in size.

TECHNICAL FIELD

The present invention relates to tissue specific anti-cancer therapeuticcompositions, to a novel method for identifying said compositions, andto methods of use of said compositions in diagnosing and treatment ofcancer.

BACKGROUND AND SUMMARY OF THE INVENTION

Prostate cancer is the most commonly diagnosed malignancy andsecond-most prevalent cause of cancer death in American men. Theestimated death of prostate cancer in 2015 was 27,540, which accountedfor about 9% of all male cancer deaths in the United States.Conventional therapies for prostate cancer include surgery, radiation,and hormone therapy. Although these treatments are relatively efficientfor early stage prostate cancer, it is known that most patients withlocalized prostate cancer ultimately relapse. Chemotherapy is currentlywidely used for advanced prostate cancer treatment, but with limitedsuccess. Lack of targeted delivery, partly because of the lack of agentsthat possess tissue specificity, is one of the major hurdles that limitthe effectiveness of cancer chemotherapy. Thus, a great deal ofattention has been paid to the development of targeted drug deliverysystems for prostate cancer therapy.

Prostate-specific membrane antigen (PSMA), also known as NAAG peptidase,glutamate carboxypeptidase II (GCPII), orN-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), is atransmembrane glycoprotein that in humans is encoded by the FOLH1(folate hydrolase 1) gene. Human PSMA contains 750 amino acids,including a large extracellular domain (707 amino acids), anintracellular domain (19 amino acids), and a transmembrane domain (24amino acids). It weighs approximately 100 kDa. It is an enzyme that is atype II transmembrane glycosylated protein with folate hydrolaseactivity, and is known to be overexpressed not only in nearly allprostate cancer cells but also in tumor neovasculature of a variety ofcancers (see: Wright Jr., G. L., et al., Urol. Oncol., 1995, 1(1):18-28;Chang, S. S., et al., Clin. Cancer Res., 1999, 5(10):2674-2681; O'Keefe,D. S., et al., Biochim. Biophys. Acta, 1998, 1443(1-2):113-127). Bycontrast, its expression in normal prostate epithelium tissues and othernormal tissues has been reported to be 100-1000 fold lower (see: WrightJr., G. L., et al., Urol. Oncol., 1995, 1(1):18-28; and others).Moreover, the expression level of PSMA correlates with prostate cancerprogression. PSMA, therefore, is a validated target for prostate cancertherapy and has been adopted as a biomarker for diagnosis and imaging,and as a targeting receptor for prostate cancer therapy because of itsoverexpression in most prostate cancer cells. Aptamers and antibodiestargeting PSMA have been discovered for targeted drug delivery toprostate cancer cells in the past few years (see: Barve, A., et al., J.Control. Release, 2014, 187:118-132). Although aptamers and antibodiesretain high binding affinity to PSMA, their drawbacks, such as largesize, possible immunogenicity and instability, may limit theirapplications in targeted drug delivery. In contrast, peptides arebelieved to have several advantages, including a small molecular weight,high permeability, great stability, less immunogenicity, ease ofsynthesis, and flexibility in chemical conjugation. Moreover, it hasbeen reported that peptides can achieve high binding affinity andspecificity that are comparable with antibodies (see: Pazgier, M., etal., Proc. Natl. Acad. Sci. U.S.A, 2009, 106(12):4665-4670; Huang, L.,et al., J. Biol. Chem., 2003, 278(18): 15532-15540).

As a carboxyl peptidase, it has been reported that PSMA can cleave theterminal glutamate from NAAG or γ-linked polyglutamate. It was alsoreported that the enzymatic activity of PSMA was found elevated inprostate cancer cells, indicating its important role in prostate cancerprogression by regulating angiogenesis. Therefore, it has been suggestedthat inhibition of the PSMA enzymatic activity could be a potentialtherapeutic approach for prostate cancer. Aggarwal and colleaguesidentified the peptide WQPDTAHHWATL (SEQ ID NO: 1), which canspecifically bind to the catalytic site of PSMA and inhibit itsenzymatic activity with an IC₅₀ of 23 μM (Aggarwal, S., et al., CancerRes., 2006, 66(18):9171-9177). However, it has been found that theGTIQPYPFSWGY (GTI) peptide (SEQ ID NO: 2) disclosed herein (see below)does not inhibit the PSMA enzymatic activity, which may be taken asindicating that the GTI peptide binds to a different site of PSMAextracellular domain (ECD) rather than the catalytic site.

Phage display has been widely used to identify peptide ligands for awide variety of molecular targets, including proteins and variousmolecular moieties, cells, or animal tissues. A phage display librarycontains billions of different phages, and each phage retains a uniqueinserted peptide sequence on the surface. Phage display technologytherefore provides a high-throughput tool for affinity selection.Protein-based biopanning and cell-based biopanning are the two mostcommon strategies to identify peptide ligands, but both of them havedisadvantages when they are used alone (see: Chen, Z., et al., Mol.Pharm., 2015, 12(6):2180-2188; Qin, B., et al., Pharm. Res., 2011,28(10):2422-2434).

Binding affinity of peptide ligands to their receptor is known to begenerally lower compared to antibodies. However, there are severalstrategies to increase the binding affinity of phage derived peptides.For example, affinity maturation is often employed to improve thebinding affinity of peptide ligands by mutagenesis. After additionalrounds of selection with these affinity maturation libraries, peptideligands with higher affinity can be discovered. In addition,dimerization or tetramerization is a common approach to improve thebinding affinity of peptides. For instance, it has been recentlydemonstrated that dimerization of an IGF2R-specific peptide improves itsapparent affinity by nearly 9-fold (see: Chen, Z., et al., Mol. Pharm.,2015, 12(6):2180-2188). Modification of peptide side-chains andsubstitution of D-amino acids are other reported strategies to improvebinding affinity. For example, Chen and colleagues replaced glycineswith D-form amino acids and significantly improved the binding affinityand stability of the peptide (see: Chen, S., et al., Chembiochem, 2013,14(11):1316-1322).

Most biopannings so far are conducted on a single target, such as arecombinant protein, a cell line, or a tissue. However, each of thesemethods has its own advantages and disadvantages. For example, arecombinant protein may exhibit a different conformation structure fromits native form in cells. Therefore, a peptide ligand discovered bybiopanning on recombinant protein may not exhibit the same affinity toits target cells in vitro and in vivo. On the other hand, the intricateand complex structures of cell membranes may lead to the discovery of apeptide ligand that binds to an unknown moiety. A recently conductedwhole cell biopanning on PSMA-positive LNCaP cells identified a peptideligand that exhibits very high affinity and specificity to LNCaP cells;however, this peptide was not PSMA-specific and its target moiety isunknown (Qin, B., et al., Pharm. Res., 2011, 28(10):2422-2434).Subsequently, future clinical application of this type of peptideligands may be unpredictable. In addition, peptide ligands identifiedfrom in vitro biopanning may not survive the complex environment in thebody after systemic administration.

In one embodiment of the invention, provided herein is a novel methodfor identifying PSMA-specific peptides that may be useful in vivo forprostate cancer diagnosis and therapy. In one aspect, said methodincludes a step of combinatorial biopanning against recombinant humanPSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaPxenografts in nude mice. Details of said novel method are providedbelow. A related embodiment of the invention herein provides novelpeptides identified by using said method, which exhibit high affinityand specificity to PSMA in vitro and in vivo. One particular peptideidentified is GTIQPYPFSWGY (or GTI) (SEQ ID NO: 2), which shows highaffinity and specificity to PSMA in vitro and in vivo, and exhibitssignificantly higher uptake in tumor tissue than uptake in othertissues, including liver, kidneys, muscle, heart, lungs, and spleen. Inanother embodiment, it is demonstrated herein that said GTI peptide canbe used in diagnosis of cancer by virtue of its ability to localize oncancer cells, such as PSMA-positive prostate cancer cells. Thus, saidGTI peptide is capable of delivering attached imaging agents to thecancer cells. Illustratively, said GTI peptide was used as describedherein to deliver an attached fluorescence agent to PSMA-positiveprostate cancer cells. In another embodiment, it is demonstrated hereinthat said GTI peptide can be used in therapy of cancer cells, such asPSMA-positive prostate cancer cells, by delivering attached therapeuticcargos to the cancer cells. Illustratively, said GTI peptide was used asdescribed herein to deliver a fused proapoptotic peptide, known to beincapable of entering the cells on its own, to PSMA-positive prostatecancer cells, resulting in cytotoxicity to the cancer cells. Thisdemonstrates that said GTI peptide mediates internalization of theproapoptotic peptide into the cells.

Another embodiment of the invention herein provides a novelcombinatorial phage biopanning procedure developed to discoverPSMA-specific peptides that can potentially be used as ligands fortargeted drug delivery to prostate cancer cells. This procedure includesconducting multiple rounds of biopanning against recombinant human PSMAextracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaPxenografts in nude mice. In one illustrative example, five rounds ofbiopanning against recombinant human PSMA extracellular domain (ECD),PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice wereconducted, and various affinity assays were carried out to identifyhigh-affinity peptides for PSMA ECD and PSMA-positive prostate cancercells. Among these high affinity peptides, the GTI peptide disclosedherein shows the highest affinity as well as specificity to PSMA inprostate cancer cells. The apparent K_(d) values of the GTI peptide toPSMA-positive LNCaP and C4-2 cells are 8.22 μM and 8.91 μM,respectively. It is disclosed herein that the GTI peptide canspecifically deliver the proapoptotic peptides to the prostate cancercells to induce cell death. One such proapoptotic peptide,_(D)(KLAKLAK)₂, fused to the GTI peptide, is successfully delivered toLNCaP cells, inducing cell death. In a biodistribution study, the GTIpeptide disclosed herein shows the highest uptake in C4-2 xenografts,while its uptake in other major organs, such as the liver and spleen,are either low or negligible. Compared to its scrambled control (randompermutation of the GTI peptide), the GTI peptide exhibits higher andmore specific uptake in C4-2 xenografts. All the results disclosedherein indicate that the GTI peptide is a potentially promising ligandfor PSMA-targeted drug delivery for prostate cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing the combinatorial phage biopanning of theinvention, which was conducted against recombinant human PSMAextracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaPxenografts in nude mice.

FIG. 2 displays the binding affinity of selected phage clones in LNCaPcells. (A) Binding of the phages on PSMA-positive LNCaP andPSMA-negative PC-3 cells that were examined using cell phage ELISA. Theratio of absorbance in LNCaP and PC-3 cells was calculated. Results arerepresented as the mean±SD (n=3). (B) Peptide sequences of the selectedphage clones (SEQ ID NOS 4, 3, 15, 2, 16, 5, 17, 6, 7, 18, 8, 19, 20, 9and 21-23, respectively, in order of appearance).

FIG. 3 shows the results of competitive inhibition of peptides and theircorresponding phages. LNCaP cells were pre-incubated with syntheticpeptides and then incubated with their corresponding phages. The boundphages were recovered and titered. (A) Competitive inhibition of eightselected peptides at 100 μM. (B) Competitive inhibition of the GTI andNRP peptide at a series of concentrations. Results are represented asthe mean±SD (n=3).

FIG. 4 shows the binding affinity of the GTI phage in LNCaP andPSMA-negative cells. The cells were suspended and incubated with the GTIphage at 4° C. for 1 h, and the bound phages were eluted and titered.The number of GTI phages that bound to LNCaP cells was normalized as100%. (A) Binding of the GTI phage and an insertless phage in LNCaPcells. (B) Binding of the GTI phage in PSMA-positive LNCaP cells andPSMA-negative cells. Results are represented as the mean±SD (n=3).(***p<0.001, **p<0.01).

FIG. 5A shows the binding affinity of the peptides NRP, SMA, GTI, andTGH in LNCaP cells. The cells were suspended and incubated with a seriesof concentrations of FAM-labeled peptides at 4° C. for 1 h. The cellswere then subjected to flow cytometry analysis to determine the percentof cells that take up the peptides. Equilibrium dissociation curves ofthe selected peptides in LNCaP cells are displayed. Results arerepresented as the mean±SD (n=3).

FIG. 5B shows the binding affinity of the peptides YPT, HSD, QPG, andYVN in LNCaP cells. The cells were treated and analyzed as describedabove for FIG. 5A. Equilibrium dissociation curves of the selectedpeptides in LNCaP are displayed.

FIG. 5C shows the binding affinity of the peptides GTI, TGH, HSD, andYVN in C4-2 cells. The cells were treated and analyzed as describedabove for FIG. 5A. Equilibrium dissociation curves of the selectedpeptides in C4-2 cells are displayed.

FIG. 5D displays the apparent equilibrium dissociation constants (K_(d))of the selected peptides in LNCaP and C4-2 cells.

FIG. 5E displays binding results of the peptides (1 μM, 10 μM and 100μM) in PC-3 cells.

FIG. 6 displays the results of the cellular uptake of the FAM-labeledGTI peptide in PSMA positive and negative cells. The FAM-labeled GTIpeptide was incubated with the cells at 37° C. for 1 h, followed bywashing with PBS and examination under a confocal microscope.

FIG. 7 displays the results of downregulating the expression of PSMA,which inhibits cellular uptake of the FAM-labeled GTI peptides in LNCaPcells. LNCaP cells were treated with 2 nM DHT before the cellular uptakestudy to downregulate the expression of PSMA. LNCaP cells without DHTtreatment were used as the control. (A) The expression of PSMA in normaland DHT treated LNCaP cells was examined using western blot. (B) LNCaPcells were incubated with 10 μM FAM-labeled GTI peptide at 37° C. for 1h and then examined under a confocal microscope. (C) Suspended LNCaPcells were incubated with FAM-labeled GTI peptide at 4° C. for 1 h,followed by flow cytometry analysis to determine cellular uptake.Results are represented as the mean±SD (n=3). (***p<0.001).

FIG. 8 shows that the GTI peptide enhances the uptake and apoptoticeffect of a proapoptotic peptide (KLA) in PSMA-positive cells. TheGTI/KLA fusion peptide, the mixture of GTI and KLA peptides, and KLApeptide were incubated with the PSMA-positive LNCaP cells (A) andPAMS-negative PC-3 cells (B) for 48 h, followed by MTT assay todetermine the apoptotic effect. The viability concentration-responsecurve of the GTI/KLA fusion peptide in LNCaP cells was presented in (C).Results are represented as the mean±SD (n=3).

FIG. 9 shows the biodistribution of the GTI peptide (SEQ ID NO: 2) andits scrambled peptide (SEQ ID NO: 13) in nude mice bearing prostatecancer xenografts. The apparent K_(d) values of the GTI peptide and itsscrambled form in C4-2 cells were determined using flow cytometry.FAM-labeled GTI and its scrambled peptides were injected into nude micebearing C4-2 xenograft tumors via the tail vein. The mice weresacrificed two hours post-administration, and major organs including thetumor, liver, kidneys, muscle, heart, lungs, and spleen were harvestedfor fluorescence imaging using a Xenogen IVIS imaging system. Data arepresented as the mean±SE (n=4). (*p<0.05).

FIG. 10A shows the binding affinity of the prostate cancer specificpeptide GTI in LNCaP cells. LNCaP cells were suspended and incubatedwith a series of concentrations of the peptides at 4° C. for 1 h,followed by flow cytometry analysis to determine cellular uptake.Results are represented as the mean±SD (n=3).

FIG. 10B shows the binding affinity of the prostate cancer specificpeptide KYL in LNCaP cells. The cells were treated and analyzed asdescribed above for FIG. 10A.

FIG. 10C shows the binding affinity of the prostate cancer specificpeptide WQP in LNCaP cells. The cells were treated and analyzed asdescribed above for FIG. 10A.

DETAILED DESCRIPTION

Before the present methods, implementations and systems are disclosedand described, it is to be understood that this invention is not limitedto specific components, specific methods, specific implementation, or toparticular compositions, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular implementations only and is not intended to belimiting. Neither are mechanisms which have been provided to assist inunderstanding the disclosure meant to be limiting.

One embodiment of the invention provides a novel method that is usefulfor identifying peptides that exhibit high affinity and specificity tocancer cells, in vitro and in vivo, particularly to PSMA-positiveprostate cancer cells. This method is represented schematically in FIG.1, and is described in detail in the Examples section. This methodcomprises peptide screening steps, including the use of a combinatorialphage biopanning technique. Illustratively, in the case of PSMA, thenovel method comprises the steps of: (a) carrying out combinatorialphage biopanning in vitro and in vivo in multiple rounds againstrecombinant human PSMA extracellular domain, PSMA-positive LNCaP cells,and LNCaP xenograft tumors; (b) randomly selecting and amplifying a setof individual phage clones from the combinatorial phage biopanning step;(c) determining the binding affinities of the selected phage clones inPSMA-positive LNCaP and PC-3 cells using cell phage ELISA; (d) selectingthe phage clones with higher affinity for PSMA-positive LNCaP cells;and, (e) sequencing the phage clones selected in the previous step toidentify the peptides expressed by these phage clones. Additionally, themethod may further comprise the steps of: (a) selecting the phage clonesthat encode the identified peptides that were found in both in vitro andin vivo biopannings; (b) pre-incubating LNCaP cells with the encodingpeptides, followed by washing, and by incubating with the correspondingphages; and, (c) identifying the peptides that show the highestcompetitive inhibition relative to their corresponding phages.Accordingly, by using the identification method described above and inthe Examples section, several PSMA-specific peptide compositions thatshow high affinity and specificity to PSMA extracellular domain (ECD)and PSMA-positive prostate cancer cells, in both in vitro and in vivoassays, were identified, including:

GTIQPYPFSWGY, (SEQ ID NO: 2) SMAGEQISWALI, (SEQ ID NO: 3) NRPDSAQFWLHH,(SEQ ID NO: 4) QPGHAILAQHPT,  (SEQ ID NO: 5) YVNSHSILGYTG,(SEQ ID NO: 6) TGHLYPTRMEIQ, (SEQ ID NO: 7) HSDNHYRPADKL, (SEQ ID NO: 8)and YPTDWLWHGHNK. (SEQ ID NO: 9)

Another embodiment of the invention provides PSMA-specific peptidecompositions identified by use of the novel method described herein.These PSMA-specific peptide compositions show high affinity andspecificity to PSMA extracellular domain (ECD) and PSMA-positiveprostate cancer cells, in both in vitro and in vivo assays.Additionally, it was discovered that these PSMA-specific peptidecompositions do not inhibit PSMA enzymatic activity. Without being boundby theory, this discovery may be taken as indicating that thePSMA-specific peptide compositions of the invention bind at a differentsite of the PSMA extracellular domain rather than the catalytic site. Inone aspect, the PSMA-positive prostate cancer cells, towards which thepeptide compositions of the invention exhibit high affinity andspecificity, include the cancer cells known as LNCaP cells, C4-2 cells,and CWR22Rv1 cells, but may also include other similar cancer cells aswell. Illustrative of the PSMA-specific peptide compositions of theinvention are the following peptides, identified by the above method:GTIQPYPFSWGY (SEQ ID NO: 2), SMAGEQISWALI (SEQ ID NO: 3), NRPDSAQFWLHH(SEQ ID NO: 4), QPGHAILAQHPT (SEQ ID NO: 5), YVNSHSILGYTG (SEQ ID NO:6), TGHLYPTRMEIQ (SEQ ID NO: 7), HSDNHYRPADKL (SEQ ID NO: 8), andYPTDWLWHGHNK (SEQ ID NO: 9). In particular, the PSMA-specific peptideGTIQPYPFSWGY (or GTI for short) (SEQ ID NO: 2) identified herein showsvery high affinity and specificity towards PSMA-positive prostate cancercells. In one key aspect of the invention, the PSMA-specific peptidecompositions herein show significantly higher uptake in tumor tissuethan in other tissues, including the liver, kidneys, muscle, heart,lungs, and spleen. For example, the PSMA-specific peptide GTI exhibitedan uptake ratio in tumor tissue relative to muscle tissue of at least3:1. This discovery of a higher uptake of the PSMA-specific peptidecompositions into tumor tissue relative to other tissues is veryinteresting, because it is well known that the kidneys and liver are themajor sites of peptide metabolism. Stated another way, this means thatthe affinity of the PSMA-specific peptide compositions, such as GTI, tothe kidneys and liver is much lower than their affinity to tumor tissue.

In another embodiment, provided herein is a diagnostic composition forcancer imaging. This diagnostic composition comprises one or more of thePSMA-specific peptide compositions identified by the method describedherein and one or more imaging agents. The one or more PSMA-specificpeptide compositions and the one or more imaging agents are fused (orattached) together using any of the fusing (or attaching) techniquesknown in the art, such as via covalent bonding, ionic bonding, and thelike, to form a diagnostic composition having a peptide portion withPSMA-specificity and affinity and an imaging moiety. A preferredembodiment of the invention is a diagnostic composition formed by fusingtogether GTI with an imaging agent. Any of the common imaging agentsknown in the art may be used, such as any of the known fluorescingagents. Illustratively, demonstrated in the examples herein is adiagnostic composition formed by combining GTI and fluorescein amidite(commonly known as FAM) to form a FAM-labeled GTI diagnosticcomposition.

In another embodiment, provided herein is a method of diagnosing cancer,such as prostate cancer, in a patient, said method utilizing thediagnostic composition described above. A related embodiment provides amethod of use of the diagnostic composition described above to diagnosecancer, such as prostate cancer, in a patient. Thus, either methodcomprises the step of administering an effective amount of thediagnostic composition to the patient, allowing sufficient time for thediagnostic composition to localize on any cancer cells, such as prostatecancer cells, and then using an imaging technique to determine thepresence or absence of cancer cells. Illustratively, the FAM-labeled GTIdiagnostic composition described above may be used. As contemplatedherein, any suitable imaging technique known in the art may be used.Also, as contemplated herein, the cancer diagnostic method herein may beused in conjunction with other cancer diagnostic methods known in theart.

In another embodiment, provided herein are ligands for targeted drugdelivery to cancer cells, such as prostate cancer cells. Said ligandscomprise a peptide that shows high affinity and specificity to cancercells. In a preferred embodiment, the ligands comprise a PSMA-specificpeptide that shows high affinity and specificity to PSMA extracellulardomain and PSMA-positive prostate cancer cells in vitro and in vivo. Inone aspect of the preferred embodiment, the peptide does not inhibitPSMA enzymatic activity. Illustrative of the PSMA-positive prostatecancer cells are LNCaP cells, C4-2 cells, or CWR22Rv1 cells, and thelike. In another important aspect, the peptide comprised in the ligandsshows higher uptake in tumor tissue than in other tissues including theliver, kidneys, muscle, heart, lungs, and spleen. For example, theuptake ratio of the peptide in tumor tissue relative to muscle tissue isat least 3:1. Illustrative of the peptides comprised in the ligands arethe following peptides, identified by the above biopanning method:GTIQPYPFSWGY (GTI) (SEQ ID NO: 2), SMAGEQISWALI (SEQ ID NO: 3),NRPDSAQFWLHH (SEQ ID NO: 4), QPGHAILAQHPT (SEQ ID NO: 5), YVNSHSILGYTG(SEQ ID NO: 6), TGHLYPTRMEIQ (SEQ ID NO: 7), HSDNHYRPADKL (SEQ ID NO:8), and YPTDWLWHGHNK (SEQ ID NO: 9). In a preferred embodiment, thepeptide comprised in the ligands is GTI.

In another embodiment, provided herein is a therapeutic composition fortreating cancer. This therapeutic composition comprises a PSMA-specificpeptide identified via the identification method described above and abiologically active moiety, wherein the PSMA-specific peptide and thebiologically active moiety are fused (or attached) to each other. Thefusing (or attaching) may be accomplished via any of the techniquesknown in the art, and may be covalent bonding, ionic bonding, and thelike. This provides a therapeutic composition having a peptide portionwith PSMA-specificity and affinity and a biologically active moiety.Illustratively, the PSMA-specific peptide may be any one of GTIQPYPFSWGY(GTI) (SEQ ID NO: 2), SMAGEQISWALI (SEQ ID NO: 3), NRPDSAQFWLHH (SEQ IDNO: 4), QPGHAILAQHPT (SEQ ID NO: 5), YVNSHSILGYTG (SEQ ID NO: 6),TGHLYPTRMEIQ (SEQ ID NO: 7), HSDNHYRPADKL (SEQ ID NO: 8), andYPTDWLWHGHNK (SEQ ID NO: 9). In a preferred embodiment, thePSMA-specific peptide is GTI. The biologically active moiety can be anycytotoxic moiety. In one illustrative example, the biologically activemoiety can be a proapoptotic moiety. For example, the proapoptoticmoiety can be one that is derived by attaching the antimicrobial peptideKLAKLAKKLAKLAK (SEQ ID NO: 10) to the PSMA-specific peptide. In a secondillustrative example, the biologically active moiety can be ananticancer moiety derived by attaching an anti-cancer drug to thePSMA-specific peptide. As contemplated herein, any anti-cancer drugknown in the art may be used.

Another embodiment of the invention provides a method of use of thetherapeutic composition described above for treating a patient sufferingfrom cancer. In a preferred embodiment, it is used for treating apatient suffering from prostate cancer. This method of use comprises thestep of administering to the patient a therapeutically effective amountof the therapeutic composition, which therapeutic composition comprisesa PSMA-specific peptide, e.g., GTI, and a biologically active moiety, asdescribed herein. Alternatively, the method comprises the step ofadministering to the patient a therapeutically effective amount of apharmaceutically or medicinally acceptable formulation or preparationthat includes the therapeutic composition of the invention. Ascontemplated herein, any of the pharmaceutically or medicinallyacceptable formulations or preparations known in the art may be used.Also, as contemplated herein, the method of use of the therapeuticcomposition herein may include co-administration with one or more otherdrugs, compositions, and/or formulations, including other anti-cancer orchemotherapeutic drugs. Likewise, the method may include, or be combinedwith, the use of any other co-therapies, such as, illustratively,radiation therapy, and the like.

While the novel technology herein has been illustrated and described indetail in the foregoing description, and in the following examples andthe figures herein, the same is to be considered as illustrative and notrestrictive in character. It is understood that one of ordinary skill inthe art could readily make a nigh-infinite number of insubstantialchanges and modifications to the above-described embodiments and that itwould be impractical to attempt to describe all such embodimentvariations in the present specification. Accordingly, it is understoodthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected.

EXAMPLES

The following examples further illustrate specific embodiments of theinvention. However, the following examples should not be interpreted inany way to limit the invention.

Example 1

Materials. The Ph.D.™-12 phage display peptide library and E. coliER2738 were purchased from New England Biolabs (Beverly, Mass.). LNCaP,PC-3, C4-2, CWR22Rv1, and HeLa cells were obtained from American TypeCulture Collection (Manassas, Va.). HSC-T6 cell line was kindly providedby Dr. Scott L. Friedman from New York University. _(D)(KLAKLAK)₂peptide was purchased from Anaspec, Inc (Fremont, Calif.). All otherpeptides including FAM-labeled peptides and GTI-KLA fusion peptides werepurchased from United Biosystems Inc (Herndon, Va.). Non-enzymatic celldissociation solution was obtained from MP Biomedicals (Santa Ana,Calif.). Homozygous nude mice were ordered from The Jackson Laboratory(Bar Harbor, Me.).

Example 2

Cell culture. LNCaP, C4-2, CWR22Rv1 and PC-3 cells were maintained inRPMI-1640 medium with 10% FBS, 100 U/mL penicillin and 100 μg/mLstreptomycin. HSC-T6 and HeLa cells were cultured in DMEM medium with10% FBS and penicillin/streptomycin. The cells were incubated in a 5%CO₂-humidified atmosphere incubator at 37° C. and passaged when theyreached 80% confluence.

Example 3

Cloning and expression of PSMA ECD. The plasmid pcDNA3.1-PSMA encodingfull-length human PSMA was provided by Dr. Shawn E. Lupold (JohnsHopkins University School of Medicine, Baltimore, Md.). PSMA ECD wasamplified from the plasmid using the forward primer5′-ATCAGATCTAAATCCTCCAATGAAGC-3′ (SEQ ID NO: 11) and reverse primer5′-ATCAAGCTTCTGCACTGTGAAGGCTGCAACATA-3′ (SEQ ID NO: 12). The amplifiedfragment was excised using BglII and HindIII, purified using a PCRClean-Up kit, and cloned into the pRSET A vector (Invitrogen, GrandIsland, N.Y.) as published (Jia, X., et al., Mol. Pharm., 2007,4(2):199-207). The pRSET A vector encoding PSMA ECD was transformed intoBL21 (DE3)pLysS competent cells. The transformed competent cells werecultured in LB medium, and IPTG was added to induce the expression ofPSMA ECD when the OD600 reached 0.5. After four hours of induction, thecells were harvested, lysed, and the expressed protein was collected.

Example 4

Combinatorial phage biopanning against the recombinant human PSMA ECD,LNCaP cells, and xenograft LNCaP tumor. As seen in FIG. 1, acombinatorial phage biopanning procedure was conducted againstrecombinant human PSMA ECD (the first, third, and fifth rounds), LNCaPcells (the second and fourth rounds), and xenograft LNCaP tumor (thefifth round). Biopanning against recombinant protein was conducted aswas published before (Chen, Z., et al., Mol. Pharm., 2015,12(6):2180-2188). Briefly, ten micrograms of the recombinant human PSMAECD was coated on 24-well plates at 4° C. overnight. Ten microliters ofthe M13 phage display library (10¹³ pfu/mL) were incubated with PSMA ECDat 4° C. for one hour under gentle shaking. Unbound phages were removedby washing the immobilized PSMA ECD with PBST (0.1% Tween 20) threetimes. Bound phages were eluted by adding 0.2 M glycine-HCl (pH 2.2) andamplified by infecting ER2738 bacteria.

Biopanning against LNCaP cells was conducted as reported (Qin, B., etal., Pharm. Res., 2011, 28(10):2422-2434; Giordano, R. J., et al., Nat.Med., 2001, 7(11):1249-1253). Briefly, PC-3 and LNCaP cells wereharvested with ice cold PBS containing 5 mM EDTA and suspended inRPMI-1640 medium at a density of 1×10⁷ cells/mL. Phages from theprevious round (10¹¹ pfu) were incubated with PC-3 cells (PSMA negative)at 4° C. for one hour to remove non-specific bound phages. Theprecleaned phages were incubated with LNCaP cells (PSMA positive) at 4°C. for one hour under gentle rotation. The cell suspension was thenmixed with 200 μL of an organic phase composed of dibutyl phthalate andcyclohexane (9:1, v/v). After centrifugation at 10,000 RPM for 10 min,the mixture was snap frozen using liquid nitrogen. The bottom of thetube was sliced off, and bound phages in the cell pellet were recoveredby infecting ER2738 bacteria.

Part of the phages from the fourth round was used for an additionalbiopanning against recombinant PSMA ECD, while the other part was usedfor in vivo biopanning against xenograft LNCaP tumor. The animalexperiment was conducted under a protocol approved by the University ofMissouri-Kansas City Institutional Animal Care and Use AdministrativeAdvisory Committee. Male nude mice aged 5-6 weeks were subcutaneouslyinoculated with LNCaP cells to generate prostate cancer xenografts. Oncethe tumor volume reached 1 cm³, 2×10⁹ pfu of the fourth round phageswere injected into the nude mouse via tail vein. After one hour, thenude mouse was sacrificed and the heart was perfused with 50 mL PBS toremove unbound phages in the body. The xenograft tumors were harvestedand homogenized in TBS buffer. After centrifugation to remove thesupernatant, the cell pellet was washed three times with TBS buffer (pH7.4), followed by the addition of 0.2 M glycine HCl (pH 2.2) to adjustthe pH to 3.0. After incubation at room temperature for 10 min, thesupernatant containing eluted phages was collected and neutralized to pH8.5 by Tris buffer (pH 9.1).

Individual phage clones from the fourth and fifth rounds of biopanningwere randomly selected, cultured and sequenced as published before(Chen, Z., et al., Mol. Pharm., 2015, 12(6):2180-2188). Encoded peptidessequences were deduced from the phage DNA sequences.

Example 5

Cell phage ELISA. LNCaP and PC-3 cells were seeded in 96-well plates ata concentration of 2×10⁴ cells per well. After 24 h, the cells werefixed with cold methanol-acetone (1:1, v/v). Phages suspended in RPMI1640 medium were incubated with the fixed cells for 1 h, followed bywashing with PBS to remove unbound phages. The cells were incubated withHRP conjugated anti-M13 monoclonal antibodies for 1 h. After adding TMBsubstrate, the absorbance at 450 nm was measured with a Beckman DTX 880multimode Detector (Beckman coulter, Inc., Brea, Calif.).

Example 6

Binding affinity to various types of cells. LNCaP, PC-3, HSC-T6, andHeLa cells were detached using nonenzymatic cell dissociation solutionand suspended in medium with 1% BSA at a density of 1×10⁷ cells/mL. Thesuspended cells were incubated with the GTI phages at 4° C. for 1 hunder gentle rotation. The cell suspension was then mixed with anorganic phase composed of dibutyl phthalate and cyclohexane (9:1, v/v).After centrifugation at 10,000 RPM for 10 min, the mixture was snapfrozen using liquid nitrogen. The bottom of the tube was sliced off, andbound phages in the cell pellet were recovered by infecting ER2738bacteria.

Example 7

Competitive inhibition assay. Competitive binding inhibition betweenselected phages and their encoded peptides was examined in this study.LNCaP cells (1×10⁷ cells/mL) suspended in RPMI-1640 medium wereincubated with encoded peptides at 4° C. for 30 min under gentlerotation. The cells were then washed, resuspended in fresh RPMI-1640medium containing phage clones, and incubated at 4° C. for 1 h. Thebound phages were recovered and titered as described above.

Example 8

Flow cytometry analysis. Cells were detached using non-enzymatic celldissociation solution and suspended in PBS (pH=7.4) at a concentrationof 1×10⁶ cells/mL. A series of concentrations (0.1-100 μM) ofFAM-labeled peptides were incubated with 500 suspended cells at 4° C.for 1 h under gentle rotation. The cells were then washed with PBS forthree times and subjected to fluorescence analysis on a FACSCalibur flowcytometer (BD Biosciences, Franklin Lakes, N.J.).

Example 9

Cellular uptake of FAM-labeled peptides Cells were seeded in 4-wellchambers at a density of 5×10⁴ cells per well and incubated at 37° C.for 24 h. The cells were then washed with PBS and then incubated with 10μM FAM-labeled peptides in Opti-MEM medium at 37° C. for 1 h. Afterincubation, the cells were gently washed three times with DPBS, fixedwith 10% formalin, and mounted with VECTASHIELD® mounting mediumcontaining DAPI. Cellular uptake of the peptides was examined using alaser scanning confocal microscope (Leica TCS SP5).

Example 10

DHT (5-a-dihydrotestosterone) treatment. LNCaP cells were cultured inPRMI-1640 medium with 5% charcoal-stripped FBS (DHT free) for 24 h.Subsequently, the cells were incubated in 5% charcoal-stripped FBSPRMI-1640 medium containing 2 nM DHT for 48 h. FAM-labeled GTI peptide(10 μM) was incubated with DHT-treated LNCaP cells and normal LNCaPcells at 37° C. for 1 h. The cells were washed, fixed with 10% formalin,and mounted with VECTASHIELD® mounting medium containing DAPI. Cellularuptake of the GTI peptide was examined using a laser scanning confocalmicroscope (Leica TCS SP5).

The expressions of PSMA in the DHT-treated LNCaP and normal LNCaP cellswere examined using western blot. Briefly, the cells were lysed on icewith RIPA buffer containing protease and phosphatase inhibitor cocktail.Protein concentrations were determined by a BCA protein assay kit.Equivalent amounts of protein (20 μg) were separated by a 12% SDS-PAGEgel. The proteins was transferred to PVDF membrane, blocked with 5%nonfat milk at room temperature for 2 h, and probed with the primaryanti-PSMA antibody (Abcam, Cambridge, Mass.). The protein was thenvisualized with horseradish peroxidase-conjugated secondary antibody andthe FluorChem FC2 imaging system (Alpha Innotech, CA).

Example 11

Biodistribution study. The animal protocol was approved by theUniversity of Missouri-Kansas City, Institutional Animal Care and UseCommittee (IACUC). The C4-2 xenograft tumor model was developed bysubcutaneous injection of 1×10⁶ C4-2 cells with matrigel into each flankof BALB/c nude male mice aged 5-6 weeks. Once the tumor is formed (˜1cm³), 20 nmol FAM-labeled GTI peptide and its scrambled peptide (randompermutation of the GTI peptide) were injected into the mice via tailvein. After two hours, the mice were sacrificed, and major organsincluding the tumors, liver, heart, spleen, lungs, kidneys, and musclewere harvested. Fluorescence intensity of these organs was examinedusing a Xenogen IVIS imaging system (Xenogen, Hopkinton, Mass.).

Example 12

Statistics analysis. Data were presented as the mean standard deviation(SD) or standard error (SE). Difference between any two groups wasevaluated by ANOVA. P<0.05 is considered statistically significant.

Example 13

Identification of PSMA-specific peptides. A novel combinatorial phagebiopanning procedure was developed to discover PSMA-specific peptidesthat can be potentially used as ligands for targeted drug delivery toprostate cancer. As shown in FIG. 1, five rounds of biopanning againstrecombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaPcells, and LNCaP xenografts in nude mice were conducted. One hundred andtwenty-two individual phage clones from the fourth and fifth rounds ofbiopanning were randomly selected and amplified. Binding affinities ofthese selected phage clones were examined in PSMA-positive LNCaP andPSMAnegative PC-3 cells using cell phage ELISA. The ratio of absorbancein LNCaP and PC-3 cells (LNCaP/PC-3) was calculated to identify phageclones that show higher binding affinity for LNCaP than PC-3 cells.Fifty phage clones with higher affinity for PSMA-positive LNCaP cellswere selected and sequenced. These phage clones express 17 differentpeptide sequences which are listed in FIG. 2.

As shown in FIG. 2A, GTI phage encoding the peptide GTIQPYPFSWGY (SEQ IDNO: 2) shows the highest binding affinity to LNCaP cells. FIG. 2B liststhe frequency of each peptide sequence in these fifty phages. The NRPphage encoding the peptide NRPDSAQFWLHH (SEQ ID NO: 4) repeats 6 timesfrom in vitro biopanning and 10 times from in vivo biopanning. Thephages encoding the peptides SMAGEQISWALI (SEQ ID NO: 3), GTIQPYPFSWGY(SEQ ID NO: 2), QPGHAILAQHPT (SEQ ID NO: 5), YVNSHSILGYTG (SEQ ID NO:6), TGHLYPTRMEIQ (SEQ ID NO: 7), HSDNHYRPADKL (SEQ ID NO: 8), andYPTDWLWHGHNK (SEQ ID NO: 9) were also found not only in in vitro butalso in vivo biopannings. Therefore, these eight phages were selectedfor further evaluation.

Competitive inhibition of selected phages and their correspondingpeptides. It was first evaluated whether these phages bind to LNCaPcells via specific interaction with their encoding peptides. LNCaP cellswere pre-incubated with the encoding peptides, followed by washing andincubation with the corresponding phages. The bound phages were theneluted and titered. As shown in FIG. 3A, the GTI peptide shows thehighest competitive inhibition to its corresponding phage. The TGH andYVN peptides exhibit relatively weaker competitive inhibition comparedto the GTI peptide, while competitive inhibition of the peptides SMA,HSD, QPG, YPT and NRP are negligible. FIG. 3B further illustrates thecompetitive inhibition effect of the GTI and NRP peptides at variousconcentrations. The GTI peptide exhibits a concentration dependentinhibition to its corresponding phage, while the NRP peptide does notaffect the binding of its corresponding phage to LNCaP cells. Thisresult may suggest that the GTI phage binds to LNCaP via the samereceptor as its peptide, while the NRP phage may bind to LNCaP via adifferent moiety as its encoding peptide.

Example 14

Binding affinity and specificity of the GTI phage to PSMA-Positive LNCaPcells. Next. the binding affinity of the GTI phage was compared toPSMA-positive LNCaP and PSMA-negative cells. Compared to insertlessphage, which does not encode a peptide, the GTI phage exhibits a muchhigher binding affinity to LNCaP cells (FIG. 4A). In addition, the GTIphage exhibits higher affinity to LNCaP cells than to PSMA-negativecells (FIG. 4B), which may further indicate that the GTI phagespecifically binds to PSMA on LNCaP cells.

Example 15

Apparent equilibrium dissociation constant (K_(d)) of the peptides. Toquantitate the apparent affinity of phage encoding peptides to prostatecancer cells, FAM-labeled peptides were incubated with PSMA-positiveLNCaP cells at different concentrations. Flow cytometry was employed todetermine the percentage of cells that were labeled with the peptides.The apparent K_(d) values were calculated by fitting to a one-sitespecific binding model using GraphPad Prism 5. As illustrated in FIG.5A, FIG. 5B, and FIG. 5D, the HSD, GTI, TGH, and YVN peptides exhibithigher binding affinity compared to other peptides. This is inaccordance with the results of their corresponding phages in FIGS. 2 and3. Their apparent K_(d) values against LNCaP cells are 12.80 μM, 8.22μM, 8.01 μM, and 9.34 μM, respectively. Also, the apparent K_(d) valuesof HSD, GTI, TGH, and YVN were examined in another PSMA-positiveprostate cancer C4-2 cell line (FIG. 5C and FIG. 5D). The GTI and YVNpeptides show similar affinity to C4-2 as to LNCaP cells, while the HSDand TGH peptides exhibit less affinity to C4-2 cells compared to LNCaPcells. Binding affinities of these FAM-labeled peptides in PC-3 cellsare presented in FIG. 5E. The GTI peptide shows the lowest bidingaffinity in PC-3, which may indicate its high specificity toPSMA-positive cells. As a result, the GTI peptide was selected as thebest PSMA-specific ligand for following studies.

Example 16

Cellular uptake of FAM-labeled GTI peptide in various PSMA-PositiveCells. Next, cellular uptake of the FAM-labeled GTI peptide wasevaluated in three PSMA-positive cells, including LNCaP, C4-2 andCWR22Rv1. PSMA-negative PC-3 cells were used as a control. Asillustrated in FIG. 6, the GTI peptide shows high uptake in all threePSMA-positive cells, while its uptake in PC-3 cells is negligible.

To determine whether cellular uptake of the GTI peptide is mediated byPSMA, the expression of PSMA in LNCaP cells was downregulated using5-a-dihydrotestosterone (DHT) (FIG. 7A) (McNamara 2^(nd), J. O., et al.,Nat. Biotechnol., 2006, 24(8):1005-1015; Israeli, R. S., et al., CancerRes., 1994, 54(7):1807-1811) and then examined the uptake of theFAM-labeled GTI peptide. As shown in the confocal images (FIG. 7B),uptake of the GTI peptide in DHT-treated LNCaP cells is much lowercompared to that in untreated LNCaP cells. Flow cytometry assay (FIG.7C) shows a similar result. The percentage of cells labeled by the GTIpeptide is significantly reduced when the expression of PSMA isdownregulated by DHT. These results clearly demonstrate that the GTIpeptide is a PSMA-specific ligand.

Example 17

The GTI Peptide Enhances the Uptake and Apoptotic Effect of aProapoptotic Peptide. The objective of this study was to identify aPSMA-specific peptide that can be used a targeting ligand to delivervarious therapeutic agents into PSMA-positive prostate cancer cells. Itwas therefore critical to demonstrate that the GTI peptide can deliver acargo to prostate cancer cells. The antimicrobial proapoptotic peptideKLAKLAKKLAKLAK (KLA) (SEQ ID NO: 10) was therefore used as a model drugin this study. Once inside cells, the KLA peptide can inducemitochondrial disruption and cellular toxicity by triggeringpermeabilization and swelling of the mitochondria. However, the KLApeptide itself cannot enter the cell. Instead, it has to be fused with aprotein transduction domain to exert its apoptotic activity (see:Javadpour, M. M., et al., J. Med. Chem., 1996, 39(16):3107-3113; Mai, J.C., et al., Cancer Res., 2001, 61(21):7709-7712). For the purpose of theinvention herein, a GTI-KLA fusion peptide was designed and itsproapoptotic activity in LNCaP and PC-3 cells was evaluated. A series ofconcentrations of the fusion peptide GTI-KLA, the mixture of GTI and KLApeptides, and the KLA peptide were incubated with LNCaP cells and PC-3cells for 48 h. As FIG. 8A indicates, the GTI-KLA fusion peptidedemonstrates cytotoxicity in LNCaP cells, whereas no cytotoxicity wasobserved in either the mixture of GTI KLA peptides or the KLA peptidealone up to 20 μM. Meanwhile, the GTI-KLA fusion peptide does not showcytotoxicity in PC-3 cells (FIG. 8B), which may indicate that the fusionpeptide enters LNCaP cells via PSMA. The IC₅₀ of the GTI-KLA fusionpeptide in LNCaP cells is approximately 12.10 μM (FIG. 8C). This studydemonstrates the potential of using the GTI peptide as a PSMA-specificligand to deliver therapeutic agents to prostate cancer cells.

Example 18

Biodistribution study. In order to evaluate the distribution profile ofthe GTI peptide in vivo, 20 nmol of FAM-labeled GTI peptide(GTIQPYPFSWGY (SEQ ID NO: 2)) and its scrambled peptide (randompermutation of the GTI peptide, SGYQTFYWPGPI (SEQ ID NO: 13)) wereinjected into nude mice bearing subcutaneous C4-2 xenograft tumor viathe tail vein. The GTI peptide exhibits lower apparent K_(d) in C4-2prostate cancer cells compared to its scrambled peptide (FIG. 9). It hasbeen reported that peptide achieves its highest uptake in tissues 2 hpost-administration (Chen, x., et al., Cancer Res., 2004,64(21):8009-8014), therefore the mice were sacrificed at 2 hpost-administration and major organs harvested including the tumor,liver, kidneys, muscle, heart, lungs, and spleen. As illustrated in FIG.9, the GTI peptide shows higher uptake in the tumors than other tissuesincluding the liver and kidneys, which are the major sites for peptidemetabolism (see: Carone, F. A., et al., Am. J. Physiol., 1980,238(3):F151-F158). Uptake of the GTI peptide in other organs, such asthe heart, lungs and muscle is negligible. Moreover, the GTI peptideexhibits much higher uptake in the tumors in comparison to its scrambledpeptide. On the contrary, the scrambled peptide shows the highest uptakein the liver. These results demonstrate that the GTI peptide canspecifically bind to PSMA overexpressing C4-2 xenografts in vivo,suggesting its potential promise as a PSMA-specific ligand for prostatecancer targeted drug delivery.

Example 19

Comparison of the GTI peptides with other prostate cancer specificPeptides. Applicants have recently discovered a peptide KYLAYPDSVHIW(KYL) (SEQ ID NO: 14) that can specifically bind to LNCaP cells (see:Qin, B., et al., Pharm. Res, 2011, 28(10):2422-2434). WQPDTAHHWATL (WQP)(SEQ ID NO: 1) is another peptide that is reported to bind to thecatalytic site of PSMA and inhibit its enzymatic activity (see:Aggarwal, S., et al., Cancer Res., 2006, 66(18):9171-9177). Therefore, acomparison was performed of the apparent binding affinity of FAM-labeledGTI, KYL, and WQP peptides to LNCaP cells. As shown in FIG. 10A, FIG.10B, and FIG. 10C, the GTI peptide exhibits the highest binding affinityto LNCaP cells with an apparent K_(d) of 8.22 μM, while the apparentK_(d) values of KYL and WQP peptides are 14.36 and 23.57 μM,respectively.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments of treatments of theconditions described herein, and the like.

As such, the particular embodiments or elements of the inventiondisclosed by the description or shown in the figures or tablesaccompanying this application are intended to be exemplary of thenumerous and varied embodiments generically encompassed by the inventionor equivalents encompassed with respect to any particular elementthereof. In addition, the specific description of a single embodiment orelement of the invention may not explicitly describe all embodiments orelements possible; many alternatives are implicitly disclosed by thedescription and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of a “treatment” shouldbe understood to encompass disclosure of the act of a “treating”,whether explicitly discussed or not; and, conversely, the disclosure ofan act of “treating”, whether explicitly discussed or not, should beunderstood to encompass disclosure of a “treatment” and even a “meansfor treating.” Such alternative terms for each element or step are to beunderstood to be implicitly included in the description.

In addition, as to each term used, it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes, for example, the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. The term “about” generally refers to a rangeof numeric values that one of skill in the art would consider equivalentto the recited numeric value or having the same function or result.Similarly, the antecedent “substantially” means largely, but not wholly,the same form, manner or degree and the particular element will have arange of configurations as a person of ordinary skill in the art wouldconsider as having the same function or result. When a particularelement is expressed as an approximation by use of the antecedent“substantially,” it will be understood that the particular element formsanother embodiment.

It is to be understood that, as used herein, the grammatical conjunction“and/or” refers throughout to either or both of the statedpossibilities.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity unless otherwiselimited. As such, the terms “a” or “an”, “one or more” and “at leastone” can be used interchangeably herein.

Also, for the purposes of the present invention, it is to be understoodthat the volume units “mL” and “cc” are considered to be approximatelyequal. As such, these units can be used interchangeably herein.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thedesired biological or medicinal response in a tissue system, animal, orhuman that is being sought by a researcher, veterinarian, medicaldoctor, or other clinician, which includes alleviation of the symptomsof the disease or condition being treated. In one aspect, thetherapeutically effective amount is that which may treat or alleviatethe disease/condition or symptoms of the disease/condition at areasonable benefit/risk ratio applicable to any medical treatment.However, it is to be understood that the total daily usage of thecompounds and compositions described herein may be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically-effective dose level for any particular patientwill depend upon a variety of factors, including the condition beingtreated and the severity of the condition; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, gender, and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctor,or other clinician of ordinary skill.

In addition, in those embodiments described herein drawn to combinationtherapy comprising administration of the therapeutic composition of theinvention and one or more other drugs or agents, “therapeuticallyeffective amount” refers to that amount of the combination of agentstaken together so that the combined effect elicits the desiredbiological or medicinal response. For example, the therapeuticallyeffective amount of the therapeutic composition of the invention and theone or more other drugs or agents, would be the total amount of thecombination of agents that when taken together or sequentially, have acombined effect that is therapeutically effective.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from variousamorphous, non-amorphous, partially crystalline, crystalline, and/orother morphological forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from varioushydrates and/or solvates of the compounds described herein. Accordingly,such compositions that recite compounds described herein are to beunderstood to include each of, or any combination of, the variousmorphological forms and/or solvate or hydrate forms of the compoundsdescribed herein.

The terms “fuse”, “fused”, and the like, as used herein to refer toattachment of one component, composition, molecule, peptide, agent, oranother entity to one or more other component, composition, molecule,peptide, agent, or another entity, are to be understood as referring toany kind of attachment known in the art that would provide sufficientstability so that the resulting attached entities can provide theintended function or use. Illustratively, fusing can be via covalentbonding, ionic bonding, or any other suitable attachment known to theskilled artisans.

The term “administering” as used herein includes all means known tothose skilled in the medical arts of introducing the compounds andcompositions described herein to the patient. It is to be understoodthat, in the methods described herein, the individual components of aco-administration, or combination, can be administered by any suitablemeans, contemporaneously, simultaneously, sequentially, separately or ina single pharmaceutical formulation. Where the co-administered compoundsor compositions are administered in separate dosage forms, the number ofdosages administered per day for each compound may be the same ordifferent. The compounds or compositions may be administered via thesame or different routes of administration. The compounds orcompositions may be administered according to simultaneous oralternating regimens, at the same or different times during the courseof the therapy, concurrently in divided or single forms.

Thus, the Applicants should be understood to claim at least: (i) aPSMA-specific peptide composition as in the Claims, and/or a method foridentifying PSMA-specific peptides as in the Claims, and/or a diagnosticcomposition for cancer imaging comprising a PSMA-specific peptide and animaging label moiety as in the Claims, and/or a method of use of thediagnostic composition as in the Claims, and/or a ligand for targeteddrug delivery to prostate cancer cells as in the Claims, and/or atherapeutic composition for treating cancer comprising a PSMA-specificpeptide and a biologically active moiety as in the Claims, and/or amethod of use of the therapeutic composition for treating a patientsuffering from prostate cancer; (ii) the related methods disclosed anddescribed; (iii) similar equivalent, and even implicit variations ofeach of these devices and methods; (iv) those alternative embodimentswhich accomplish each of the functions shown, disclosed, or described;(v) those alternative designs and methods which accomplish each of thefunctions shown as are implicit to accomplish that which is disclosedand described; (vi) each feature, component, and step shown as separateand independent inventions; (vii) the applications enhanced by thevarious systems or components disclosed; (viii) the resulting productsproduced by such systems or components; (ix) methods and apparatusessubstantially as described hereinbefore and with reference to any of theaccompanying examples; (x) the various combinations and permutations ofeach of the previous elements disclosed.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed, or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification are hereby incorporated byreference as part of this description of the invention, and theapplicants expressly reserve the right to use all of or a portion ofsuch incorporated content of such claims as additional description tosupport any of or all of the claims or any element or component thereof,and the applicants further expressly reserve the right to move anyportion of or all of the incorporated content of such claims or anyelement or component thereof from the description into the claims orvice-versa as necessary to define the matter for which protection issought by this application or by any subsequent application orcontinuation, division, or continuation-in-part application thereof, orto obtain any benefit of reduction in fees pursuant to, or to complywith the patent laws, rules, or regulations of any country or treaty,and such content incorporated by reference shall survive during theentire pendency of this application including any subsequentcontinuation, division, or continuation-in-part application thereof orany reissue or extension thereon.

Additionally, the claims set forth in this specification are furtherintended to describe the metes and bounds of a limited number of thepreferred embodiments of the invention and are not to be construed asthe broadest embodiment of the invention or a complete listing ofembodiments of the invention that may be claimed. The applicants do notwaive any right to develop further claims based upon the description setforth above as a part of any continuation, division, orcontinuation-in-part, or similar application.

While the disclosure has been illustrated and described in detail in thefigures and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly selected embodiments have been shown and described and that allchanges, modifications and equivalents that come within the spirit ofthe disclosures described heretofore and/or defined by the followingclaims are desired to be protected. It will be apparent to one ofordinary skill in the art that various changes and modifications can bemade to the claimed invention without departing from the spirit andscope thereof. Thus, for example, those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. In addition, all publications cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety as if each had beenindividually incorporated by reference and fully set forth.

What is claimed is:
 1. A peptide consisting of the amino acid sequenceof SEQ ID NO:
 2. 2. The peptide of claim 1, wherein said peptidespecifically binds to the extracellular domain of human PSMA.
 3. Thepeptide of claim 1, wherein said peptide specifically binds to theextracellular domain of human PSMA at the surface of a cell and isinternalized by the cell.
 4. The peptide of claim 3, wherein said cellis a prostate cancer cell that overexpresses human PSMA.
 5. The peptideof claim 1, wherein the peptide shows higher uptake in tumor tissueexpressing human PSMA than in other tissues selected from tissues of theliver, kidney, muscle, heart, lung, and spleen.
 6. The peptide of claim5, wherein the uptake ratio in tumor tissue expressing human PSMArelative to muscle tissue is at least 3:1.
 7. A composition comprisingthe peptide of claim
 1. 8. A composition comprising the peptide of claim1 fused to an imaging agent.
 9. The composition of claim 8, wherein saidimaging agent is a fluorescent agent.
 10. The composition of claim 8,wherein said fluorescent agent is fluorescein amidite.
 11. A compositioncomprising the peptide of claim 1 fused to a therapeutic agent.
 12. Thecomposition of claim 11, wherein said therapeutic agent is ananti-cancer or chemotherapeutic drug.